四环素抑制系统调节新生隐球菌必需基因表达的适应性。

IF 3.7 2区 生物学 Q2 MICROBIOLOGY
mSphere Pub Date : 2025-05-27 Epub Date: 2025-05-01 DOI:10.1128/msphere.01018-24
Ci Fu, Nicole Robbins, Leah E Cowen
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引用次数: 0

摘要

机会性人类真菌病原体新隐球菌作为隐球菌脑膜炎的病原体对人类健康有巨大的影响,迫切需要扩大我们目前的抗真菌武器库。基本基因产物通常是抗菌剂的理想靶点,识别和表征感兴趣的病原体中的基本基因对药物开发至关重要。不幸的是,由于C. neoformans的单倍体性质和缺乏产生有效条件表达突变体的遗传工具,对其必需基因的表征受到限制。迄今为止,铜抑制启动子pCTR4是最广泛使用的调节必需基因表达的系统;但其表达是漏性的,铜具有多效性。在多种真菌物种中,包括酿酒酵母菌、白色念珠菌和耳念珠菌,四环素抑制启动子系统是调控基因表达的有力工具;然而,它还没有适应新的C.。在这项研究中,我们成功地在新生C. neoformans中实施了四环素抑制系统来调节必需基因HSP90和FKS1的表达。在培养物中添加四环素类似物强西环素可以有效地减少HSP90转录物和蛋白质水平,并抑制新生C.的生长和活力。同样,用强力霉素去除FKS1增强了菌株对棘球白菌素caspofungin的敏感性,这是一种针对葡聚糖合成酶的抗真菌药物,但通常对新生C.无效。因此,这项工作揭示了一种新的方法来产生条件表达突变的C.新生,提供了前所未有的潜力,系统地研究重要的人类真菌病原体的基本基因功能。侵袭性真菌感染每年导致数百万人死亡,而可用于对抗这些病原体的抗真菌药物的数量仅限于三类:多烯类、偶氮类和棘白菌素。新型抗真菌药物靶点的最大来源是必需的基因产物,这是细胞生存所必需的。然而,鉴定和表征新生C.必需基因的工具是非常有限的。在这里,我们采用了四环素抑制启动子系统,该系统已广泛应用于其他生物中,以研究C.新生生物的必需基因功能。通过将这个可调节启动子置于必需基因HSP90和FKS1的上游,我们证实了菌株在四环素类似物多西环素存在下的生长导致必需基因表达的缺失。该方法为系统地研究新生C.必需基因提供了重要的进展。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Adaptation of the tetracycline-repressible system for modulating the expression of essential genes in Cryptococcus neoformans.

The opportunistic human fungal pathogen Cryptococcus neoformans has an enormous impact on human health as the causative agent of cryptococcal meningitis, and there is a dire need to expand our current antifungal arsenal. Essential gene products often serve as ideal targets for antimicrobials, and identifying and characterizing essential genes in a pathogen of interest is critical for drug development. Unfortunately, characterization of essential genes in C. neoformans is limited due to its haploid nature and lack of genetic tools for generating effective conditional-expression mutants. To date, the copper-repressible promoter pCTR4 is the most widely used system to regulate essential gene expression; however, its expression is leaky and copper has pleiotropic effects. In diverse fungal species, including Saccharomyces cerevisiae, Candida albicans, and Candida auris, the tetracycline-repressible promoter system is a powerful tool to regulate gene expression; however, it has yet to be adapted for C. neoformans. In this study, we successfully implemented the tetracycline-repressible system in C. neoformans to regulate the expression of the essential genes HSP90 and FKS1. Supplementation of cultures with the tetracycline analog doxycycline efficiently depleted HSP90 at both transcript and protein levels and inhibited C. neoformans growth and viability. Similarly, the depletion of FKS1 with doxycycline enhanced sensitivity of the strain to the echinocandin caspofungin, an antifungal that targets the glucan synthase but is generally ineffective against C. neoformans. Thus, this work unveils a novel approach to generate conditional-expression mutants in C. neoformans, providing unprecedented potential to systematically study essential gene function in this important human fungal pathogen.IMPORTANCEInvasive fungal infections cause millions of deaths annually, while the number of antifungals available to combat these pathogens is limited to only three classes: polyenes, azoles, and echinocandins. The largest source of novel antifungal drug targets are essential gene products, which are required for cellular viability. However, tools to identify and characterize essential genes in C. neoformans are extremely limited. Here, we adapted the tetracycline-repressible promoter system, that has been widely used in other organisms, to study essential gene function in C. neoformans. By placing this regulatable promoter upstream of the essential genes HSP90 and FKS1, we confirmed that the growth of the strains in the presence of the tetracycline analog doxycycline results in the depletion of essential gene expression. This approach provides a significant advance for the systematic study of essential genes in C. neoformans.

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来源期刊
mSphere
mSphere Immunology and Microbiology-Microbiology
CiteScore
8.50
自引率
2.10%
发文量
192
审稿时长
11 weeks
期刊介绍: mSphere™ is a multi-disciplinary open-access journal that will focus on rapid publication of fundamental contributions to our understanding of microbiology. Its scope will reflect the immense range of fields within the microbial sciences, creating new opportunities for researchers to share findings that are transforming our understanding of human health and disease, ecosystems, neuroscience, agriculture, energy production, climate change, evolution, biogeochemical cycling, and food and drug production. Submissions will be encouraged of all high-quality work that makes fundamental contributions to our understanding of microbiology. mSphere™ will provide streamlined decisions, while carrying on ASM''s tradition for rigorous peer review.
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